LOCUS       sequence-496184-        4497 bp    DNA     circular SYN 05-JAN-2026
DEFINITION  Plasmid contains gRNA scaffold-tRNA and is used to amplify fragments
            for Golden Gate assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4497)
  AUTHORS   Kozgunova E
  TITLE     tRNA-Based Polycistronic CRISPR/Cas9 System Boosts Efficiency of 
            Multi-Gene Deletion in the Moss Physcomitrella
  JOURNAL   bioRxiv 2025.07.29.667574
REFERENCE   2  (bases 1 to 4497)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 5, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.07.29.667574"
COMMENT     Sequence Label: EKv376(gRNAscaffold-tRNA for cloning)
FEATURES             Location/Qualifiers
     source          1..4497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        751..826
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        907..953
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     CDS             975..2000
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    2042..2218
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(2276..2292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2276..2292)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2289..2311)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2300..2316
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2324..2330,2331..2348,2349..2354))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2369..2390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2507..2524)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2678..3266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2758..2777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3437..4228,4229..4297))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4060..4079
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4298..4402)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4470..4488
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcac aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttcgattg aatgtccatt
      421 gaagcagacg tgttgcgaca ggttagcgac gatgggtgta gatgtgatgt gatgtgatgg
      481 tgtggttctt ccacggcggc gtccttgcgg tggcggagaa ggggatatcc cgaaggagcg
      541 gcagcgggag agcacaagca gaaagggtgc agtgagtgag tgggtccagc tgggtggctg
      601 gccgagtgga cgcgaccggg tttcgagggg gggggggaga aaagggatgg agcgagggat
      661 ataacccaca tggaatggag gtgggtgtga aggcgggtat ataggaaggt ggaggactta
      721 caacccatgc aattgcacgc tttaatcgat gttttagagc tagaaatagc aagttaaaat
      781 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcaaca aagcaccagt
      841 ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt
      901 gcatctgcaa gacccttcct ctatataagg aagttcattt catttggaga ggacagccca
      961 agcaaggagg tgatatgaag aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     1021 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     1081 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     1141 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     1201 aagtgcttga cattggagag ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     1261 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctt cagccggtcg
     1321 cggaggctat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     1381 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     1441 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     1501 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     1561 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     1621 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     1681 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     1741 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     1801 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     1861 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     1921 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     1981 gtcctagggc aaaggaatag tgaaatcacc agtctctctc tacaaatcta tctctctcta
     2041 ttttctccat aaataatgtg tgagtagttt cccgataagg gaaattaggg ttcttatagg
     2101 gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat ttgtaaaata
     2161 cttctatcaa taaaatttct aattcctaaa accaaaatcc agtactaaaa tccagatctc
     2221 ctaaagtccc tattctagag gatccccggg taccgagctc gaattcgtaa tcatggtcat
     2281 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2341 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2401 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2461 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2521 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2581 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2641 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2701 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2761 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2821 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2881 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2941 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3001 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3061 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3121 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3181 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3241 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3301 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3361 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3421 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3481 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3541 gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca ccggctccag
     3601 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3661 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3721 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3781 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3841 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3901 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3961 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4021 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4081 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4141 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4201 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4261 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4321 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4381 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4441 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
